Immunohistochemical studies have revealed that calcitonin gene- related peptide (CGRP) coexists with acetylcholine in motor cells of the spinal cord. Therefore this study was undertaken to investigate a possible interaction between the cholinergic nerve neurotransmitter and CGRP on neuromuscular transmission in the isolated rat diaphragm. Electrical stimulation of the isolated phrenic nerve resulted in twitch contractions which were dose- dependently potentiated by CGRP in concentrations ranging from 1.2 X 10-9 M to 3 X 10-7 M. The potentiating action of CGRP (3 X 10-7 M) returned to control level in about 25 min and was rarely tachyphylactic. The action of CGRP was dependent upon the stimulation pulse width ranging from 0.2 to 1.0 msec. Rat calcitonin (4.5 X 10-7 M) caused a minimal change in the amplitude of twitch contractions. CGRP had no effect on the quiescent striated muscle. Twitch responses to direct electrical stimulation was also enhanced by CGRP (6 X 10-8 M - 6 X 10-7 M) in the absence and presence of 10-5 M d-tubocurarine. These results suggest that CGRP modulates the action of acetylcholine at the motor-end plates of striated muscle. The possibility that an alteration in the normal peptide content in nerve endings (motor-end plates) of the body may lead to a variety of muscle malfunctions is of great clinical significance and should be studied in humans.